1. Glycoprotein O-Linked Man Type Sugar Chain
Among proteins derived from eukaryotes, glycoproteins modified with sugar chains are frequently found rather than simple proteins composed of only amino acids. It is known that these sugar chains are responsible for the stability of proteins or the maintenance of tertiary structure, or they play important roles in intermolecular recognition for intercellular adhesion, etc. Major sugar chains of glycoproteins include the N-linked type binding to asparagine and O-linked type binding to serine (Ser) or threonine (Thr) (Makoto Takeuchi, Glycobiology Series 5, Glycotechnology, edited by Akira Kihata, Sen-ichiro Hakomori, and Katsutaka Nagai, Kodansha Scientific, 191–208 (1994)). In all the cases regarding N-linked type, N-acetylglucosamine (GlcNAc) is bound to an amide group of asparagine. On the other hand, regarding O-linked type, there exist several different kinds which have different sugars bound to the hydroxyl groups of Ser or Thr. Among these, a series of sugar chains in which mannose is bound to the amino acid are classified as the O-linked Man type. In addition to this, mucin type in which N-acetylgalactosamine (GalNAc) is bound and O-linked GlcNAc type in which N-acetylglucosamine (GlcNAc) is bound are widely known. These O-linked type sugar chains differ not only in sugars bound to amino acids but also in their entire sugar chain structures [Van den Steen, P., Rudd, P. M., Dwek, R. A., and Opdenakker, G, Crit. Rev. Biochem. Mol. Biol., (1998), 33, 151–208].
O-linked Man type sugar chains are often found in glycoproteins of yeasts, fungi, or the like. In the case of yeasts, most constituent sugars are mannose, but there are some known cases wherein a small number of phosphates or galactoses (Gal) are bound thereto. The structure of sugar chains is not-uniform and varies depending on the type of yeast. For example, a structure having 7 sugars or less, wherein 1 to 3 residues of mannose are linearly bound to a protein and additional mannose, galactose, or mannose phosphate is transferred thereto [Gemmill, T. R. and Trimble, R. B., Biochim. Biophys. Acta, (1999), 1426, 227–237].
Regarding sugar chain biosynthesis, researches have been made with a focus on baker's yeasts (Saccharomyces cerevisiae, hereinafter referred to as S. cerevisiae), and the biosynthesis is considered to proceed along the pathway as shown in FIG. 1. First, a reaction occurs wherein mannose is transferred via α-linkage to a hydroxide group of Ser or Thr with a protein O-mannosyltransferase (PMTp) [Strahl-Bolsinger, S., Gentzsch, M., and Tanner, W., Biochim. Biophys. Acta, (1999), 1426, 297–307]. Further, another mannose is bound via α-1,2 linkage with any one of KRE2p, KTR1p, and KTR3p, thereby forming a structure having 2 residues of mannose bound. When KRE2p acts on this sugar chain continuously, the structure having 3 residues of mannose bound via α-1,2 linkage is formed [Lussier, M., Sdicu, A. M., Bussereau, F., Jacquet, M., and Bussey, H., J. Biol. Chem., (1997), 272, 15527–15531]. The subsequent biosynthesis pathway is divided into two: one path wherein further 1 to 2 residues of mannose are bound thereto via α-1,3 linkage with MNN1p, MNT2p, and MNT3p [Romero, P. A., Lussier, M., Veronneau, S., Sdicu, A. M., Herscovics, A., and Bussey, H., Glycobiology, (1999), 9, 1045–1051]; and the other path wherein mannose phosphate is transferred with MNN6p [Jars, M. U., Osborn, S., Forstrom, J., and MacKay, V. L., J. Biol. Chem., (1995), 270, 24810–24817]. Meanwhile, when mannose is transferred via α-1,3 linkage, mannose phosphate transfer reaction is inhibited [Nakayama, K., Feng, Y., Tanaka, A., and Jigami, Y., Biochim. Biophys. Acta, (1998), 1425, 255–262]
In contrast, animal O-linked Man type sugar chains had been virtually unknown, but in recent years there have been reports thereon one after another. First, NeuAcα2→3Galβ1→4GlcNAcβ1→2Manα1→Ser/Thr structure was discovered from α-dystroglycan of bovine peripheral nerve [Chiba, A., Matsumura, K., Yamada, H., Inazu, T., Shimizu, T., Kusunoki, S., Kanazawa, I., Kobata, A., and Endo, T., J. Biol. Chem., (1997), 272, 2156–2162]. Thereafter, the existence of similar sugar chain structures in α-dystroglycan derived from rabbit skeletal muscles or sheep brains was confirmed [Sasaki, T., Yamada, H., Matsumura, K., Shimizu, T., Kobata, A., and Endo, T., Biochem. Biophys. Acta, (1998), 1425, 599–606, Smalheiser, N. R., Haslam, S. M., Sutton-Smith, M., Morris, H. R., and Dell, A., J. Biol. Chem., (1998), 273, 23698–23703]. Further, from rabbit brain extract, HSO3→3GlcAβ1→3Galβ1→4GlcNAcβ1→2Manα1→Ser/Thr structure which contains HNK-1 epitope that is considered to play an important role in constructing neural circuits, was reported [Yuen, C.-T., Chai, W., Loveless, R. W., Lawson, A. M., Margolis, T., and Feizi, T., J. Biol. Chem., (1997), 272, 8924–8931]. According to these reports, it is clear that O-linked Man type sugar chain also exists in animals. However, animal sugar chains have sialic acid (NeuAc), galactose, N-acetylglucosamine, glucuronic acid (GlcA), and sulfate groups bound thereto, and thus they are quite different in structure from those known in yeasts or fungi.
Biosynthesis pathway of O-linked Man type sugar chains in animals have hardly been learned. Regarding an enzyme that transfers mannose to a protein, a homolog of yeast PMT gene has been obtained, but its function has not been confirmed [Perez Jurado, L. A., Coloma, A., and Cruces, J., Genomics, (1999), 58, 171–180]. In addition, it has been discovered that the gene mutation which causes rotated abdomen in Drosophila occurs in a gene having a high homology with a yeast PMT gene [Martin-Blanco, E. and Garcia-Bellido, A., Proc. Natl. Acad. Sci. USA, (1996), 93, 6048–6052]. However, it is not clear whether this gene has PMT activity or not. Further, the enzymes which are engaged in the elongation of O-linked Man type sugar chain, such as the enzyme (OMGnT) that transfers β 1,2-linked N-acetylglucosamine to mannose which is bound to a protein, are not well-understood.
2. Function of O-Linked Man Type Sugar Chain
S. cerevisiae has 7 types of PMTp isozymes as enzymes to transfer mannose to proteins. Among these, the disruption of 3 genes (PMT1, PMT2, and PMT4 or PMT2, PMT3, and PMT4) disables its growth [Strahl-Bolsinger, S., Gentzsch, M., and Tanner, W., Biochim. Biophys. Acta, (1999), 1426, 297–307]. Further, the mutation of Drosophila rt gene, which has a high homology with the PMT gene of yeast, is a cause for morphological abnormalities [Martin-Blanco, E. and Garcia-Bellido, A., Proc. Natl. Acad. Sci. USA, (1996), 93, 6048–6052]. Therefore, there is considered to be a possibility that O-linked Man type sugar chains of proteins have important roles in the growth and morphogenesis of organisms.
It has been known that an O-linked Man type sugar chain is bound to α-dystroglycan in animals [Endo, T., Biochim. Biophys. Acta, (1999), 1473, 237–246]. α-dystroglycan along with β-dystroglycan is a glycoprotein encoded by a dystroglycan gene, and, through posttranslational cleavage, two components, α and β are formed [Ibraghimov-Beskrovnaya, O., Ervasti, J. M., Leveille, C. J., Salughter, C. A., Sernett, S. W., and Campbell, K. P., Nature, (1992), 355, 696–702]. Both of them are expressed in the basal membrane of nerve tissues or muscles, and α-dystroglycan exists with binding to an extracellular domain of β-dystroglycan which is a transmembrane protein. Further, α-dystroglycan is extracellularly bound to laminin-1, laminin-2, agrin, or other extracellular matrices, and β-dystroglycan is intracellularly bound to dystrophin or other similar cytoskeletal proteins. Therefore, α- and β-dystroglycans are assumed to play roles in joining a cytoskeleton to an extracellular matrices [Henry, M. D. and Campbell, K. P., Curr. Opin. Cell Biol., (1999), 11, 602–607]. This complicated structure wherein such a plurality of molecules are bound together is referred to as a dystrophin glycoprotein complex (DGC), and its close relation to the morphogenesis of organisms has been proven. For example, gene abnormalities of any component of dystrophin [Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C., and Kunkel, L. M., Cell, (1987), 50, 509–517], laminin-2 [Xu, H., Wu, X. R., Wewer, U. M., and Engval, E., Nature Genet., (1994), 8, 297–302] and dystroglycan [Cote, P. D., Moukhles, H., Lindenbaum, M., and Carbonetto, S., Nature Genet., (1999), 23, 338–342] cause muscular dystrophy which is a serious disease.
It is indicated that, in the linkage between α-dystroglycan and laminin in a DGC, a 3′-sialyl N-acetyllactosamine (NeuAcα2→3Galβ1→4GlcNAc) structure of the O-linked type sugar chain existing in α-dystroglycan is important [Chiba, A., Matsumura, K., Yamada, H., Inazu, T., Shimizu, T., Kusunoki, S., Kanazawa, I., Kobata, A., and Endo, T., J. Biol. Chem., (1997), 272, 2156–2162]. This sugar chain structure is substantially recognized in O-linked Man type sugar chains of skeletal muscle-derived α-dystroglycan [Sasaki, T., Yamada, H., Matsumura, K., Shimizu, T., Kobata, A., and Endo, T., Biochem. Biophys. Acta, (1998), 1425, 599–606]. Thus, there is a high likelihood that O-linked Man type sugar chains have an important role in the linkage of α-dystroglycan and laminin. If it is true, a mutation of OMGnT that is essential to the biosynthesis of this sugar chain is presumed to cause abnormalities in the DGC structure, and furthermore it is considered that OMGnT is likely to be a causative gene for yet to be elucidated muscular dystrophy-like nervous diseases or morphological abnormalities.
Accordingly, the use of antibodies/antiserum obtained by using OMGnT protein of the present invention as antigens, or the use of all or a part of the polynucleotide encoding OMGnT of the present invention as a probe is useful for the detection and genetic diagnosis of lesions, or the like caused by the above-mentioned muscular dystrophy-like nervous diseases or morphological abnormalities.
It is reported that α-dystroglycan is involved in the infection of peripheral nerve Schwann cells with the bacteria, Mycobacterium leprae (hereinafter, referred to as M.leprae), which is known to be a cause of Hansen's disease [Rambukkana, A., Yamada, H., Zanazzi, G., Mathus, T., Salzer, J. L., Yurchenco, P. D., Campbell, K. P., and Fischetti, V. A., Science, (1998), 282, 2076–2079]. The authors revealed that M. leprae is bound to the G domain of the laminin-2 α2 chain (LNα2G) that is normally bound to α-dystroglycan specifically, and selectively infects peripheral nerves. Further, M. leprae is bound via LNα2G to α-dystroglycan prepared from peripheral nerve or skeletal muscle, though such linkage is not observed in the case of recombinant α-dystroglycan prepared from E. coli, indicating that the α-dystroglycan sugar chain is an important element in the infectivity of M. leprae. 
Moreover, it is revealed that α-dystroglycan is an infection target of pathogenic viruses such as Arena viruses including the Lassa fever virus, or Lymphocytic choriomeningitis virus [Cao, W., Henry, M. D., Borrow, P., Yamada, H., Elder, J. H., Ravkov, E. V., Nichol, S. T., Compans, R. W., Campbell, K. P., and Oldstone, B. A., Science, (1998), 282, 2079–2081]. In this case, the virus is bound to α-dystroglycans derived from rabbit skeletal muscle, but not to recombinant α-dystroglycans produced by E. coli, indicating that the α-dystroglycan sugar chain has an important role in infection. Further, the virus is not bound to the α2 subunit of a dihydropyridine receptor complex which is a glycoprotein likewise derived from rabbit skeletal muscle. Therefore, it is pointed out that the sugar chain structure specifically bound to α-dystroglycan is important in the infection. Accordingly, it is considered that there is an extremely high possibility that the very rarely observed O-linked Man type sugar chain is a target of the virus.
As described above, there is a high likelihood that O-linked Man type sugar chains of α-dystroglycan are extensively involved in the infection of pathogenic bacteria or viruses. Hence, when a carbohydrate containing O-linked Man type sugar chain is administered to an organism, it is expected to suppress the binding of bacteria or virus to α-dystroglycan. This action is useful in preventing the infectivity of the above-mentioned pathogenic bacteria or viruses, or to cure infected patients and stop the deterioration of symptoms. However, in order to implement these events, large volumes of carbohydrates containing O-linked Man type sugar chains are necessary, and the use of OMGnT of the present invention for the synthesis of the carbohydrate enables this implementation. In addition, among host cells having polynucleotides encoding OMGnT introduced therein, for example, yeast cells (the details are described below), are useful since they can produce human-type O-linked Man type sugar chains at low cost.
3. Production of Mammal-Derived Glycoproteins in Yeast
Various yeasts as typified by S. cerevisiae are often used as hosts to conduct mass production of recombinant proteins. Most physiologically active mammalian proteins are obtained from living bodies usually only in extremely small amounts, and thus if inexpensive mass production of recombinant proteins with yeast is possible, this would be very useful. However, mammalian proteins are bound to sugar chains in most cases, and their structure is largely different from that of yeasts. Thus, it is often the case that the proteins simply expressed in yeasts cannot be used as pharmaceuticals [Romanos, M. A., Scorer, C. A., and Clare, J. J., Yeast, (1992), 8, 423–488, Eckart, M. R. and Bussineau, C. M., Curr. Opin. Biotechnol., (1996), 7, 525–530]. Particularly, the fact that Manα1→3Manα1→2 structure contained in yeast sugar chains has antigenicity to humans is a major problem [Young, M., Davies, M. J., Bailey, D., Gradwell, M. J., Smestad-Paulsen, B., Wold, J. K., Barnes, R. M. R., and Hounsell, E. F., Glycoconj. J., (1998), 15, 815–822].
In order to overcome these problems, in recent years, technologies to convert a yeast sugar chain structures to those similar to mammals' have been in the process of development. For example, a technology to convert yeast N-linked type sugar chain to an animal sugar chain structure has been already established [Chiba, Y., Suzuki, M., Yoshida, S., Yoshida, A., Ikenaga, H., Takeuchi, M., Jigami, Y., and Ichishima, E., J. Biol. Chem., (1998), 273, 26298–26304]. As for O-linked type sugar chain, the other major sugar chain, however, there has not been such an idea nor technology. If a technology to convert O-linked type sugar chain to a sugar chain structure similar to mammals' is established, it is considered that together with the conversion technologies for N-linked type sugar chains, problems concerning almost all the sugar chains would be solved.
4. N-acetylglucosaminyltransferase (GnT)
There have been no reports on an enzyme (OMGnT) which transfers N-acetylglucosamine via β1,2 linkage to mannose bound to serine or threonine in a protein. However, as the enzyme N-acetylglucosaminyltransferase (GnT) which transfers N-acetylglucosamine to mannose, GnT-I, GnT-II, GnT-III, GnT-IV, GnT-V, and GnT-VI have been reported [Schachter, H., Brockhausen, I., and Hull, E., Methods Enzymol., (1989), 179, 351–397]. All of these are involved in the biosynthesis of N-glycan, and each recognizes their intrinsic substrates for transfer reaction. Hitherto, cDNA cloning has been carried out for the following: GnT-I [Kumar, R., Yang, J., Larsen, R. D. and Stanley P., Proc. Natl. Acad. Sci. USA, (1990), 87, 9948–9952, Sarkar, M., Hull, E., Nishikawa, Y., Simpson, R. J., Moritz, R. L., Dunn, R., and Schachter, H., Proc. Natl. Acad. Sci. USA, (1991), 88, 234–238]; GnT-II [D'Agostaro, G A., Zingoni, A., Moritz, R L., Simpson, R J., Schachter, H. and Bendiak, B., J. Biol. Chem., (1995), 270, 15211–15221]; GnT-III [Nishikawa, A., Ihara, Y., Hatakeyama, M., Kangawa, K. and Taniguchi, N., J. Biol. Chem., (1992), 267, 18199–18204]; GnT-IV; and GnT-V [Shorebah, M. G., Hindsgaul, O. and Pierce, M., J. Biol. Chem., (1992), 267, 2920–2927]. With respect to GnT-IV, the existence of the following two isozymes has been revealed: -IVa [Yoshida, A., Minowa, M. T., Takamatsu, S., Hara, T., Oguri, S., Ikenaga, H. and Takeuchi, M., Glycobiology, (1999), 9, 303–310] and -IVb [Yoshida, A., Minowa, M. T., Takamatsu, S., Hara, T., Ikenaga, H. and Takeuchi, M., Glycoconj. J., (1998), 15, 1115–1123]. Among these, 62% amino acid sequence identity has been recognized between human GnT-IVa and GnT-IVb, which have the same enzymatic activity. However, although they catalyze similar enzymatic reactions, there exists no clear homology between GnT-I and GnT-II that transfer GlcNAc via β1,2 linkage to mannose, or between GnT-III and GnT-IV that transfer GlcNAc via β1,4 linkage to mannose. Further, any significant homology cannot be found between GnTs which differ in transfer manner of GlcNAc. From these findings, it is generally considered that there is no sequence homology among various GnTs which transfer N-acetylglucosamine to mannose.
Herein, “identity” is an expression illustrating the degree of identical individual amino acid residues constituting sequences. In this case, the expression includes a case wherein alignment processing is performed in consideration of the existence of a gap. Further, “homology” used herein is an expression whose scope includes the scope of the above “identity” and further similar amino acid usage (e.g., isoleucine and leucine, and asparagine and glutamic acid), and the like.
An object of the present invention is to provide: a protein (enzyme) having novel N-acetylglucosaminyltransferase (hereinafter referred to as OMGnT) activity; a polynucleotide encoding the above enzyme; a recombinant polynucleotide containing the above polynucleotide; cells of mammals, yeasts, or the like containing the above recombinant polynucleotide; a method for producing an enzyme protein having OMGnT activity by cultivating the cells in a medium and the enzyme protein produced by the method; a method for producing a novel substance using OMGnT; a method for producing O-linked Man type sugar chains and carbohydrates (glycosylated amino acids, glycopeptides, glycoproteins, and derivatives thereof) by culturing cells having the above polynucleotides introduced thereinto in a medium; and O-linked Man type sugar chains and carbohydrates (glycosylated amino acids, glycopeptides, glycoproteins, and derivatives thereof) produced by the above method.